Schizophrenia is a severe psychiatric disorder affecting approximately 1% of the world's population. Clinical symptoms of schizophrenia include delusions, auditory hallucinations, disorganized thoughts and speech, social withdrawal, lack of motivation, and cognitive dysfunction such as disorganized thinking and memory impairments. This disorder is believed to be caused by a combination of neurological defects including dopamine and serotonin levels, and inhibitory interneuron deficiencies. Schizophrenia can be treated with drugs which target neurotransmitters and receptors, commonly referred to as antipsychotic or neuroleptic drugs.
One class of antipsychotic drug termed “atypical antipsychotics” or more commonly “second generation antipsychotics” includes the tricyclic dibenzazepine derivative, clozapine (I). Clozapine, marketed under Clozaril® by Novartis in the United States, interferes with the binding of dopamine at the dopamine receptors D1, D2, D3, and D5, and has a high affinity for the D4 receptor (Package Insert Clozaril 2009). Clozaril also acts as an antagonist at adrenergic, cholinergic, histaminergic, and serotonergic receptors. Clozapine is almost completely metabolized by cytochrome P450 in humans to the N-desmethyl, and N-Oxide derivatives. The N-desmethyl metabolite has only limited activity, while the N-Oxide derivative is inactive. The activity of N-desmethyl metabolite is with receptors other than the receptors with which clozapine is active.
Clozapine has the following formula:

N-desmethylclozapine has the following formula:

Clozapine-N-oxide has the following formula:

Plasma concentration of clozapine is effected by varying cytochrome P450 activity (Chetty, M and M Murray, Curr Drug Metab, 8, 4, 307-13 2007; Mauri, M C, et al., Clin Pharmacokinet, 46, 5, 359-88 2007; Mendoza, M C and J P Lindenmayer, Clin Neuropharmacol, 32, 3, 154-7 2009), age, sex, caffeine use, and smoking (Rostami-Hodjegan, A, et al., J Clin Psychopharmacol, 24, 1, 70-8 2004).
Clozapine has been shown to have high inter-patient variability in plasma steady-state concentrations and this variability can impact safety and quality of life (Mauri, M C, L S Volonteri, A Colasanti, A Fiorentini, I F De Gaspari and S R Bareggi, Clin Pharmacokinet, 46, 5, 359-88 2007).
Since efficacy of clozapine is improved within a specific range of plasma drug concentration and the drug exhibits wide intra-patient pharmacokinetic variability monitoring concentrations of this drug in blood and adjusting to target levels would be of value in increasing efficacy and minimizing toxicity (Mauri, M C, L S Volonteri, A Colasanti, A Fiorentini, I F De Gaspari and S R Bareggi, Clin Pharmacokinet, 46, 5, 359-88 2007)
As a result of this variability, equal doses of the same drug in different individuals can result in dramatically different clinical outcomes. The effectiveness of the same dosage of clozapine varies significantly based upon individual drug clearance and the ultimate serum drug concentration in the patient. Therapeutic drug management would provide the clinician with insight on patient variation in drug administration. With therapeutic drug management, drug dosages could be individualized to the patient, and the chances of effectively treating the disorder without the unwanted side effects would be much higher.
In addition, therapeutic drug management of clozapine would serve as an excellent tool to ensure compliance (Treur, M, et al., BMC Health Serv. Res., 9, 9 2009; Valenstein, M, et al., J. Clin. Psychiatry, 67, 10, 1542-1550 2006) with administration and prescribed dosage to achieve effective serum concentration levels. Routine therapeutic drug management of clozapine would require the availability of simple automated tests adaptable to general laboratory equipment. The use of liquid chromatography (LC) with ultraviolet (UV) or mass spectrometry detection to determine the concentration of clozapine in human blood and plasma has been described (Rosland, M, et al., Drug Dev Ind Pharm, 33, 10, 1158-66 2007; Rao, L V, et al., J Clin Lab Anal, 23, 6, 394-8 2009; Ming, D S and J Heathcote, J Anal Toxicol, 33, 4, 198-203 2009; Niederlander, H A, et al., J Chromatogr B Analyt Technol Biomed Life Sci, 834, 1-2, 98-107 2006). These methods are labor intensive, requiring liquid-liquid or solid phase extractions, use expensive equipment and are not amenable to routine clinical laboratory use. To date, there are no immunoassays for measuring clozapine in human biological fluids of patients treated with this antipsychotic agent.
As seen from the foregoing, there are no immunoassays for determining the presence and/or quantifying the amount of clozapine in human biological fluids. Routine therapeutic drug management of clozapine by immunoassays would provide simple automated tests adapted to standard laboratory equipment. However, in order to provide such immunoassays, antibodies specific to clozapine must be produced. The derivatives and immunogen used in this assay must impart through these corresponding antibodies produced specific reactivity to clozapine without any substantial cross-reactivity reactivity to therapeutically or pharmacologically active or inactive metabolites of clozapine which interfere with the detection of clozapine, these interfering metabolites being N-desmethylclozapine, (the compound of formula II) and clozapine-N-oxide (the compound of formula III). In conducting immunoassays for monitoring clozapine, it is important that the antibodies used not be reactive with the pharmaceutically active metabolite, N-desmethylclozapine (the compound of formula II). This is true since reactivity with N-desmethylclozapine interferes with the detection of clozapine in the patient sample and there is no correlation between the detectable N-desmethylclozapine and clinical outcome. See Y.-Q. Xiang et al. Serum Concentrations of clozapine and norclozapine in the prediction of relapse of patients with schizophrenia, Schizophrenia Research, 83 (2006) 201—and M. C. Mendoza & J. P. Lindenmayer. N-Desmethylclozapine: Is there evidence for its antipsychotic potential?, Clinical Neuropharmacology 32 (3) 154-157
Therefore, in order to be effective in monitoring drug levels by means of an immunoassay, the antibodies should be specific to clozapine and not cross react with its major pharmaceutically active metabolite, N-desmethylclozapine.